Nematodes are microscopic roundworms that feed on the roots, leaves and stems of more than 2,000 row crops, vegetables, fruits, and ornamental plants, causing an estimated $100 billion crop loss worldwide. A variety of parasitic nematode species infect crop plants, including root-knot nematodes (RKN), cyst- and lesion-forming nematodes. Root-knot nematodes, which are characterized by causing root gall formation at feeding sites, have a relatively broad host range and are therefore parasitic on a large number of crop species. The cyst- and lesion-forming nematode species have a more limited host range, but still cause considerable losses in susceptible crops.
Parasitic nematodes are present throughout the United States, with the greatest concentrations occurring in the warm, humid regions of the South and West and in sandy soils. Soybean cyst nematode (Heterodera glycines), the most serious pest of soybean plants, was first discovered in the United States in North Carolina in 1954. Some areas are so heavily infested by soybean cyst nematode (SCN) that soybean production is no longer economically possible without control measures. Although soybean is the major economic crop attacked by SCN, SCN parasitizes some fifty hosts in total, including field crops, vegetables, ornamentals, and weeds.
Signs of nematode damage include stunting and yellowing of leaves, and wilting of the plants during hot periods. Nematode infestation, however, can cause significant yield losses without any obvious above-ground disease symptoms. The primary causes of yield reduction are due to underground root damage. Roots infected by SCN are dwarfed or stunted. Nematode infestation also can decrease the number of nitrogen-fixing nodules on the roots, and may make the roots more susceptible to attacks by other soil-borne plant nematodes.
The nematode life cycle has three major stages: egg, juvenile, and adult. The life cycle varies between species of nematodes. The life cycle of SCN is similar to the life cycles of other plant parasitic nematodes. The SCN life cycle can usually be completed in 24 to 30 days under optimum conditions, whereas other species can take as long as a year, or longer, to complete the life cycle. When temperature and moisture levels become favorable in the spring, worm-shaped juveniles hatch from eggs in the soil. Only nematodes in the juvenile developmental stage are capable of infecting soybean roots.
After penetrating soybean roots, SCN juveniles move through the root until they contact vascular tissue, at which time they stop migrating and begin to feed. With a stylet, the nematode injects secretions that modify certain root cells and transform them into specialized feeding sites. The root cells are morphologically transformed into large multinucleate syncytia (or giant cells in the case of RKN), which are used as a source of nutrients for the nematodes. The actively feeding nematodes thus steal essential nutrients from the plant resulting in yield loss. As female nematodes feed, they swell and eventually become so large that their bodies break through the root tissue and are exposed on the surface of the root.
After a period of feeding, male SCN migrate out of the root into the soil and fertilize the enlarged adult females. The males then die, while the females remain attached to the root system and continue to feed. The eggs in the swollen females begin developing, initially in a mass or egg sac outside the body, and then later within the nematode body cavity. Eventually the entire adult female body cavity is filled with eggs, and the nematode dies. It is the egg-filled body of the dead female that is referred to as the cyst. Cysts eventually dislodge and are found free in the soil. The walls of the cyst become very tough, providing excellent protection for the approximately 200 to 400 eggs contained within. SCN eggs survive within the cyst until proper hatching conditions occur. Although many of the eggs may hatch within the first year, many also will survive within the protective cysts for several years.
A nematode can move through the soil only a few inches per year on its own power. However, nematode infestation can spread substantial distances in a variety of ways. Anything that can move infested soil is capable of spreading the infestation, including farm machinery, vehicles and tools, wind, water, animals, and farm workers. Seed sized particles of soil often contaminate harvested seed. Consequently, nematode infestation can be spread when contaminated seed from infested fields is planted in non-infested fields. There is even evidence that certain nematode species can be spread by birds. Only some of these causes can be prevented.
Traditional practices for managing nematode infestation include: maintaining proper soil nutrients and soil pH levels in nematode-infested land; controlling other plant diseases, as well as insect and weed pests; using sanitation practices such as plowing, planting, and cultivating of nematode-infested fields only after working non-infested fields; cleaning equipment thoroughly with high pressure water or steam after working in infested fields; not using seed grown on infested land for planting non-infested fields unless the seed has been properly cleaned; rotating infested fields and alternating host crops with non-host crops; using nematicides; and planting resistant plant varieties.
Methods have been proposed for the genetic transformation of plants in order to confer increased resistance to plant parasitic nematodes. For example, a number of approaches involve transformation of plants with double-stranded RNA capable of inhibiting essential nematode genes. Other agricultural biotechnology approaches propose to over-express genes that encode proteins that are toxic to nematodes. U.S. Pat. Nos. 5,589,622 and 5,824,876 are directed to the identification of plant genes expressed specifically in or adjacent to the feeding site of the plant after attachment by the nematode.
US 2009/0089896 discloses a promoter of an Mtn21-like gene which is induced in syncytia of SCN-infected soybean. WO 2008/077892 discloses a promoter of a peroxidase-like gene which is induced in syncytia of SCN-infected soybean. WO 2008/071726 discloses a promoter of a trehalose-6-phosphate phosphatase-like gen which is induced in syncytia of SCN-infected soybean. WO 2008/095887 discloses a promoter of an Mtn3-like gene which is induced in syncytia of SCN-infected soybean. WO 2008/095888 discloses the promoter of an At5g12170-like gene which is induced in syncytia of SON-infected soybean.
A number of patent publications prophetically disclose and generically claim transgenic plants comprising any one or more of thousands of plant genes and having improved agronomic characteristics. Examples of such publications include US2004/0031072, US2006/0107345, US2004/0034888, US2004/0019927, US2004/0045049, US2004/0019927, US2006/0272060, WO2005/5112608, US2006/0150283, and US2007/0214517. Pathogen resistance, including nematode resistance, is disclosed as one potential improved agronomic characteristic of the transgenic plants described in these publications. However, none of these publications specifically associate any disclosed gene with improved nematode resistance in transgenic plants containing the gene.
Serine-Arginine rich (SR-rich) proteins are key regulators of plant gene expression, with various gene family members contributing to constitutive splicing of RNA, nuclear export, maintenance of mRNA stability and protein translation. SR proteins are also involved in alternative RNA splicing, where they bind specific RNA sequences and guide the formation of spliceosome complexes at weak splicing sites SR rich gene families are moderately populated in plants, with diverse sub-groups falling into approximately five motif-based categories.
The Avr9-elicited 111B-like gene is a transcription factor with sequence homology to 111B ACRE (Avr9/Cf-9 rapidly elicited) from Nicotiana tabacum and DREB1A/CBF3 from Arabidopsis. In tobacco the 111B ACRE gene is a pathogenesis-related transcriptional activator that is rapidly induced in lines expressing the Cf-9 resistance gene in response to Avr9 expressed by Cladosporium fulvum, a biotrophic fungus. In other species, CBF3/DREB1 genes are involved in activating abiotic stress response. U.S. Pat. No. 7,345,217 discloses SEQ ID NO:1408, an Avr9-elicited 111B-like gene which is purported to be a homolog of an Arabidopsis thaliana DNA designated G912. U.S. Pat. No. 7,345,217 generically discloses numerous categories of potential utilities for the thousands of genes disclosed therein, and one of those categories is identified as disease resistance, including nematode resistance. However, the only specific utilities proposed in U.S. Pat. No. 7,345,217 for G912 and its homologs are improved tolerances to cold, freezing, drought, and salt stress.
Basic Helix-Loop-Helix (bHLH) and Dehydration Responsive Element Binding (DREB) transcription factors are also key regulatory molecules in plants. The physiological functions of some bHLH genes have been demonstrated experimentally in plants. The R and TT8 genes are known to regulate anthocyanin accumulation in maize and Arabidopsis, and other bHLH genes interact with phytochrome and regulate light response. Other bHLH genes regulate hormone signaling. The physiological role of most plant bHLH genes is unknown, however, and there is little sequence conservation between bHLH gene family members outside of the core bHLH signature domain.
Dirigent-like proteins belong to a large, diverse gene family found in all major land plant groups analyzed to date. Dirigent-encoding genes cluster into 5 phylogenetic subfamilies, Dir-A through Dir-E. The Dir-A subfamily has been shown, in conjunction with phenolic oxidases, to direct the stereospecific assembly of lignins (cell wall components) and lignans (plant antioxidants and defense compounds) in a range of plant species. Expression of PsDIR1, a Dir-A gene from Pisum sativa, confers resistance to multiple fungal pathogens in transgenic canola. Dir-A subfamily genes are induced by a wide variety of stresses, such as mechanical wounding, herbivory and fungal infection. The specific biochemical functions of genes from subgroups Dir-B, Dir-C, Dir-D and Dir-E (Dir-like) proteins are not as well characterized, although genes from the Dir-C subfamily were shown to be induced by jasmonic acid treatment, salicylic acid and feeding by avirulent Hessian fly larvae.
To date, no genetically modified plant comprising a transgene capable of conferring nematode resistance has been deregulated in any country. Accordingly, a need continues to exist to identify safe and effective compositions and methods for controlling plant parasitic nematodes using agricultural biotechnology.